Human Liver Sinusoidal Microvascular Endothelial Cells AEQ-GFP in Mitochondria (HLiSMVECs-AEQ-GFP)

I'm excited to share with you some amazing information about our fantastic product:

Product Name: Human Liver Sinusoidal Microvascular Endothelial Cells Expressing AEQ-GFP in Mitochondria (HLiSMVECs-AEQ-GFP)
- Catalog Number: UBP011AEQ-GFP
- Product Format: Available in Frozen Vial or T-25 Flask
- Cell Number: Each vial contains more than 5x10^5 cells
- Description: Our HLiSMVECs-AEQ-GFP cells are an incredible subpopulation of puromycin resistant HLiSMVECs, derived from normal human liver tissue, and obtained after infection with AEQUOPORIN-GFP-expressing lentiviruses targeted to mitochondria. These cells are shipped in frozen vials at passage 2. For optimal culture, we recommend using Universal Endothelial Growth Medium (EGM) supplemented with 10% serum and growth factors. With proper cultivation, these cells exhibit a minimum average population doubling level greater than 16.

- Cell Characterization:
    1. Cytoplasmic VWF/Factor VIII: Immunofluorescence positive in more than 95% of cells.
    2. Cytoplasmic uptake of Di-I-Ac-LDL: Immunofluorescence positive in more than 95% of cells.
    3. Cytoplasmic PECAM1: Immunofluorescence positive in more than 95% of cells.
    4. HLiSMVECs-AEQ-GFP cells test negative for HIV-1, HBV, HCV, and mycoplasma.

- Product Usage: These incredible cells are intended for Research Use Only.

- Shipping: Our cells are shipped in Frozen Vials in Dry Ice package.

- Handling of Arriving Cells: Once you receive the dry ice package containing the frozen vials, we recommend promptly transferring the vials to a -80°C freezer for short-term storage or a liquid nitrogen tank for long-term storage.

I hope you find this information helpful, and I'm here to assist with any further questions you may have.

Cell culture instructions for a T-25 flask:

1. Ensure that the flask is at 90% confluence. If not, remove the transport media and add 5 ml of fresh media to the flask. Place the flask in a 37°C incubator until the cells reach 90% confluence, changing the media every 2 days.

2. If the flask is at 90% confluence, remove the transport media from the flask.

3. Rinse the T-25 flask containing cells with 5 ml 1X PBS.

4. Gently aspirate out the 1X PBS after rinsing and discard.

5. Add 2 ml of room temperature Universal Detachment solution to the T-25 flask containing cells, ensuring that the entire interior surface is covered.

6. Place the T-25 flask containing cells into a 37°C incubator for 1 or 2 minutes. Cells will normally come off the surface within 1 or 2 minutes.

7. Suspend the cells with 15 ml of Universal ENDO-Growth Medium and transfer them equally into 3 pre-coated T-25 flasks (the cells are now at a subculture ratio of 1:3).

8. There is no need to spin cells during subculture when Universal Cell Detachment solution is used.

9. For proliferating cell culture, change the Universal ENDO-Growth medium every 2 days. The cells normally become confluent within 7 days when split at a 1:3 ratio.

10. Use ENDO-Basal media containing 0.5% FBS to induce quiescent cells (after 18-24 hours).

Welcome to the Thawing and Subculture Protocols to ensure optimal cell culture techniques and successful experiments.

A) Pre-coating of T-25 flasks:

Prepare the T-25 flask for cell culture by applying 2ml of Universal Coating Solution, ensuring full coverage. After 5 minutes, carefully remove any excess coating solution and rinse the flask twice with 5ml of 1x PBS. Your flask is now primed and ready for use.

B) Thawing the cells:

Thaw the frozen cell vial in a 37°C water bath, leaving a small amount of ice in the vial. Then, transfer the cells into the pre-coated T-25 flask with 10ml of growth medium. Usually, the cells will reach confluency overnight and be ready for passage within a few days.

C) Subculturing the cells:

Start by rinsing the cells in a T-25 flask with 5ml of room-temperature 1xPBS, followed by adding 2ml of room-temperature Universal Detachment Solution into the flask. Gently dispose of the excess solution within 20 seconds by aspiration.

D) Detaching the cells:

Leave the T-25 flask with the cells in a 37°C incubator for 1 minute. Most cells usually detach from the surface within 1-2 minutes. Alternatively, you can monitor the cells under a microscope until most of them become rounded up. Then gently tap the flask against the bench surface, and the cells will move on the surface of the flask when observed under a microscope.

E) Centrifugation:

Add 5ml of Universal Neutralization Buffer and spin down the cells with an 800g centrifugation for 5 minutes.

F) Re-suspending the cells:

Re-suspend the cell pellet with 10 or 15ml of Universal Growth Medium and transfer 5ml each into 2 or 3 pre-coated T25 flasks for a 1/2 to 1/3 subculture ratio.

G) Maintenance:

Ensure the medium is changed every 2 or 3 days, and the cells usually reach confluency within 7 days when split at a 1/3 ratio.

H) Preparation for experiments:

For experiments, to prepare quiescent cells, replace with Universal Endothelial Basal Medium containing 0.5% FBS when the cells are nearly confluent for about 8-12 hours before your experiments.

These protocols are designed to support the growth and health of your cells, ensuring the success of your important research.